Microfluidic Synthesis of Scalable Layer-by-Layer Multiple Antigen Nano-Delivery Platform for SARS-CoV-2 Vaccines

Abstract

The COVID-19 outbreak was a global pandemic with wide-ranging healthcare implications. Although several mRNA-based vaccines delivered using lipid nanoparticles (LNP) have been approved and demonstrated efficacy at reducing the severity and spread of infection, continued rapid viral evolution and disadvantages currently associated with LNP delivery vehicles (such as toxicity) are driving the design of next-generation SARS-CoV-2 vaccines. Herein, we describe the development of a trimethylated chitosan-based nanoparticle layer-by-layer (LbL) delivery platform for multiple antigens as a scalable and safe COVID-19 vaccine, known as, "LbL-CoV19". These vaccine candidates have been demonstrated to be biocompatible, safe, and effective at stimulating both humoral and cellular responses for protection in preclinical studies. Preliminary results also indicate that LbL-CoV19 can potentially achieve rapid, long-lasting, and broad protection against the SARS-CoV-2 challenge. The "plug-and-play" platform technology is well suited to preparedness for future pandemics and disease outbreaks.

Keywords: SARS-CoV-2; T-cells; adjuvant; chitosan nanoparticle; layer-by-layer (LbL); sustained releases.

Figure 1

Scalable plug-and-play Layer-by-Layer (LbL) trimethylated chitosan NP platform for loading of SARS-CoV-2 antigens including Spike protein/peptide (SP) and Non-structural protein/peptide (NSP) as a novel vaccine candidate, (LbL-CoV19). (A) The loaded antigens in each layer could be one or two antigens or open-reading frame accessory protein/peptides (ORF) or one antigen with an adjuvant 7DW8-5; (B) The LBL sustained releases profile of each antigen has been demonstrated in vaccination for potential long-term protection.

Figure 2

Scanning electron microscopy images of TMC nanoparticles (A) and LbL-CoV-1 formulation nanoparticles (B). The inserts in the right bottom corner images are high-magnification images for TMC nanoparticles and LbL-CoV-1 formulation nanoparticles.

Figure 3

NSP Peptide 22 and Spike protein (SP) release profiles over two weeks from 1-step (solid line) and 2-step (dot line) synthesized two-layer LbL-CoV19 vaccine formulation (LbL-CoV-1). Mean (dots) ± SEM is represented for each of the above graphs.

Figure 4

The lbL-CoV19 vaccine candidate was filled in an amber bottle with different scales/doses by lyophilization and then sealed with a rubber stopper and crimped with an aluminum cap to be ready for reconstitution using saline.

Figure 5

ELISpot immunogenicity studies for LbL-CoV19 formulations. The number of IFN-γ-secreting cells was measured by ELISpot assay. The stimulation index was calculated as the number of spots detected in the respective peptide stimulated well divided by the number of spots in the media-only well, and the bars displayed are the mean of 5 animals per dose group. Samples were run in duplicate. **** indicates p < 0.0001; ** indicates p < 0.05 significant between two groups of vaccine formulations.

Figure 6

ICS studies of human CD8+ T cells (A) and CD4+ T cells (B) induced by the LbL-CoV19 vaccine candidates as determined by single cell-based multiplexed assay. The bar displays each mean ± SEM for each of the above graphs. *** indicates p < 0.005, ** indicates p < 0.05 significant between each group to control.

Figure 7

Co-encapsulation of adjuvant 7DW8-5 with peptide in formulation LbL-CoV-1.

Figure 8

ELISpot of immunized mice CD4+ and CD8+ T cell responses from one dose of LbL peptide Spike formulation with (LbL-CoV-1a) and without (LbL-CoV-1) adjuvant 7DW8-5. The bar displays each mean ± SEM for each of the above graphs. *** indicates p < 0.005, ** indicates p < 0.05 significant between two group of vaccine candidate.

Figure 9

Virus MA 10 strain TCID50 titers were determined in Lung tissue after 3 days of viral challenge for the saline mice group and four LbL-CoV1, 1a, 4, 4a vaccine groups. Each dot data point represented a mouse (blue) and an average group of mice (red) dot with STDEV of log TCID/lung weight. The dotted line indicates the limit of quantitation of the viral load. *** indicates p < 0.001 significant between each group to saline control.